The N-terminal amino group of [Tyr(8)]bradykinin is bound adjacent to analogous amino acids of the human and rat B-2 receptor

To obtain data of the bradykinin B-2 receptor's agonist binding site, Ne used a combined approach of affinity labeling and ''immunoidentification'' of receptor fragments generated by cyanogen bromide cleavage. Domain-specific antibodies to the various extracellular receptor domains mere applied to detect receptor fragments with covalently attached [I-125-Tyr(8)]bradykinin, As a cross-linker we used the homobifunctional reagent disuccinimidyl tartarate (DST), which reacts preferentially with primary amines, With this technique a [I-125-Tyr(8)]bradykinin-labeled receptor fragment derived from the third extracellular domain was identified. The epsilon-amino group of lysine (Lys(172)) of the human B-2 receptor provides the only primary amino group within this receptor fragment. This strongly suggests that DST attached the N-terminal amino group of [Tyr(8)]bradykinin to Lys(172) of the human B-2 receptor, Next me asked whether DST attaches [Tyr(8)]bradykinin to the analogous residue, Lys(174) of the rat B-2 receptor, which is 81% identical to the human B-2 receptor, and we attempted to label the wild-type rat B-2 receptor and a rat B-2 receptor mutant where Lys(174) had been exchanged for alanine. Affinity labeling of the wild-type rat B-2 receptor worked efficiently, whereas DST did not attach detectable amounts of [I-125-Tyr(8)]bradykinin to the K174A rat B-2 receptor mutant, Taken together these observations indicate that the N-terminal amino group of [Tyr(8)]bradykinin is bound to analogous positions of the rat and of the human B-2 receptor, i.e. [Tyr(8)]bradykinin's N terminus is bound adjacent to Lys(172) of the human and Lys(174) of the rat B-2 receptor.